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Insects exhibit remarkable adaptability in their locomotive strategies in diverse environments, a crucial trait for foraging, survival, and predator avoidance. Microvelia americana, tiny 2-3 mm insects that adeptly walk on water surfaces, exemplify this adaptability by using the alternating tripod gait in both aquatic and terrestrial terrains. These insects commonly inhabit low-flow ponds and streams cluttered with natural debris like leaves, twigs, and duckweed. Using high-speed imaging and pose-estimation software, we analyze M. americana movement on water, sandpaper (simulating land), and varying duckweed densities (10%, 25%, and 50% coverage). Our results reveal M. americana maintain consistent joint angles and strides of their upper and hind legs across all duckweed coverages, mirroring those seen on sandpaper. Microvelia americana adjust the stride length of their middle legs based on the amount of duckweed present, decreasing with increased duckweed coverage and at 50% duckweed coverage, their middle legs' strides closely mimic their strides on sandpaper. Notably, M. americana achieve speeds up to 56 body lengths per second on the deformable surface of water, nearly double those observed on sandpaper and duckweed, which are rough, heterogeneous surfaces. This study highlights M. americana's ecological adaptability, setting the stage for advancements in amphibious robotics that emulate their unique tripod gait for navigating complex terrains., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Premise: Polyploidy is a widespread mutational process in angiosperms that may alter population performance of not only plants but also their interacting species. Yet, knowledge of whether polyploidy affects plant-herbivore dynamics is scarce. Here, we tested whether aphid herbivores exhibit preference for diploid or neopolyploid plants, whether polyploidy impacts plant and herbivore performance, and whether these interactions depend on the plant genetic background., Methods: Using independently synthesized neotetraploid strains paired with their diploid progenitors of greater duckweed (Spirodela polyrhiza), we evaluated the effect of neopolyploidy on duckweed's interaction with the water-lily aphid (Rhopalosiphum nymphaeae). Using paired-choice experiments, we evaluated feeding preference of the herbivore. We then evaluated the consequences of polyploidy on aphid and plant performance by measuring population growth over multiple generations., Results: Aphids preferred neopolyploids when plants were provided at equal abundances but not at equal surface areas, suggesting the role of plant population surface area in driving this preference. Additionally, neopolyploidy increased aphid population performance, but this result was dependent on the plant's genetic lineage. Lastly, the impact of herbivory on neopolyploid vs. diploid duckweed varied greatly with genetic lineage, where neopolyploids appeared to be variably tolerant compared to diploids, sometimes mirroring the effect on herbivore performance., Conclusions: By experimentally testing the impacts of polyploidy on trophic species interactions, we showed that polyploidization can impact the preference and performance of herbivores on their plant hosts. These results have significant implications for the establishment and persistence of plants and herbivores in the face of plant polyploidy., (© 2024 The Authors. American Journal of Botany published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

Competing Interests: Conflict of interest statement. None declared.

Airspace or aerenchyma is crucial for plant development and acclimation to stresses such as hypoxia, drought, and nutritional deficiency. Although ethylene-mediated signaling cascades are known to regulate aerenchyma formation in stems and roots under hypoxic conditions, the precise mechanisms remain unclear. Moreover, the cellular dynamics underlying airspace formation in shoots are poorly understood. We investigated the stage-dependent structural dynamics of shoot aerenchyma in greater duckweed (Spirodela polyrhiza), a fast-growing aquatic herb with well-developed aerenchyma in its floating fronds. Using X-ray micro-computed tomography and histological analysis, we showed that the spatial framework of aerenchyma is established before frond volume increases, driven by cell division and expansion. The substomatal cavity connecting aerenchyma to stomata formed via programmed cell death (PCD) and was closely associated with guard cell development. Additionally, transcriptome analysis and pharmacological studies revealed that the organization of aerenchyma in greater duckweed is determined by the interplay between PCD and proliferation. This balance is governed by spatiotemporal regulation of phytohormone signaling involving ethylene, abscisic acid, and salicylic acid. Overall, our study reveals the structural dynamics and phytohormonal regulation underlying aerenchyma development in duckweed, improving our understanding of how plants establish distinct architectural arrangements. These insights hold the potential for wide-ranging application, not only in comprehending aerenchyma formation across various plant species but also in understanding how airspaces are formed within the leaves of terrestrial plants., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

When subjected to dietary caloric restriction (CR), individual animals often outlive well-fed conspecifics. Here, we address whether CR also extends lifespan in plants. Whereas caloric intake in animals comes from ingestion, in plants it derives from photosynthesis. Thus, factors that reduce photosynthesis, such as reduced light intensity, can induce CR. In two lab experiments investigating the aquatic macrophyte Lemna minor, we tracked hundreds of individuals longitudinally, with light intensity-and hence, CR-manipulated using neutral-density filters. In both experiments, CR dramatically increased lifespan through a process of temporal scaling. Moreover, the magnitude of lifespan extension accorded with the assumptions that (a) light intensity positively relates to photosynthesis following Michaelis-Menten kinetics, and (b) photosynthesis negatively relates to lifespan via a power law. Our results emphasize that CR-mediated lifespan extension applies to autotrophs as well as heterotrophs, and suggest that variation in light intensity has quantitatively predictable effects on plant aging trajectories., (© 2024 The Author(s). Ecology Letters published by John Wiley & Sons Ltd.)

The inflorescence (spadix) of skunk cabbage (Symplocarpus renifolius) is strongly thermogenic and can regulate its temperature at around 23 °C even when the ambient temperature drops below freezing. To elucidate the mechanisms underlying developmentally controlled thermogenesis and thermoregulation in skunk cabbage, we conducted a comprehensive transcriptome and metabolome analysis across 3 developmental stages of spadix development. Our RNA-seq analysis revealed distinct groups of expressed genes, with selenium-binding protein 1/methanethiol oxidase (SBP1/MTO) exhibiting the highest levels in thermogenic florets. Notably, the expression of alternative oxidase (AOX) was consistently high from the prethermogenic stage through the thermogenic stage in the florets. Metabolome analysis showed that alterations in nucleotide levels correspond with the developmentally controlled and tissue-specific thermogenesis of skunk cabbage, evident by a substantial increase in AMP levels in thermogenic florets. Our study also reveals that hydrogen sulfide, a product of SBP1/MTO, inhibits cytochrome c oxidase (COX)-mediated mitochondrial respiration, while AOX-mediated respiration remains relatively unaffected. Specifically, at lower temperatures, the inhibitory effect of hydrogen sulfide on COX-mediated respiration increases, promoting a shift toward the dominance of AOX-mediated respiration. Finally, despite the differential regulation of genes and metabolites throughout spadix development, we observed a convergence of gene expression and metabolite accumulation patterns during thermogenesis. This synchrony may play a key role in developmentally regulated thermogenesis. Moreover, such convergence during the thermogenic stage in the spadix may provide a solid molecular basis for thermoregulation in skunk cabbage., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Plant microbiomes that comprise diverse microorganisms, including prokaryotes, eukaryotes and viruses, are the key determinants of plant population dynamics and ecosystem function. Despite their importance, little is known about how species interactions (especially trophic interactions) between microbes from different domains modify the importance of microbiomes for plant hosts and ecosystems. Using the common duckweed Lemna minor , we experimentally examined the effects of predation (by bacterivorous protists) and parasitism (by bacteriophages) within microbiomes on plant population size and ecosystem phosphorus removal. Our results revealed that the addition of predators increased plant population size and phosphorus removal, whereas the addition of parasites showed the opposite pattern. The structural equation modelling further pointed out that predation and parasitism affected plant population size and ecosystem function via distinct mechanisms that were both mediated by microbiomes. Our results highlight the importance of understanding microbial trophic interactions for predicting the outcomes and ecosystem impacts of plant-microbiome symbiosis.

Lemna aequinoctialis Welw. is a widely spread species that has diverse physiological and molecular properties. Flower characteristics are important factors in deducing taxonomical status; however, owing to the rarity of flowering observations in Lemna, studying them has been a prolonged challenge. In this study, physiological and morphological analyses were conducted by inducing flowering, and molecular analysis was done based on the two chloroplast DNA loci (matK, atpF-atpH intergeneric spacer) of L. aequinoctialis sensu Landolt (1986) from 70 strains found in 70 localities in Japan, Korea, Thailand, and the US. In total, 752 flowering fronds from 13 strains were observed based on axenic conditions. Two different trends in flower organ development-protogyny and adichogamy-were detected in these strains. Their physiological traits were divided into two groups, showing different morphological features based on frond thickness, root cap, and anther sizes. Molecular analysis showed two lineages corresponding to two physiological groups. These were identified as L. aequinoctialis sensu Beppu et al. (1985) and L. aoukikusa Beppu et Murata based on the description of the nomenclature of L. aoukikusa. These were concluded as independent taxa and can be treated as different species. Furthermore, the distribution of L. aoukikusa is not only limited to Japan., (© 2024. The Author(s) under exclusive licence to The Botanical Society of Japan.)

Fluorescent materials and technologies have become widely used in scientific research, and due to the ability to convert light wavelengths, their application to photosynthetic organisms can affect their development by altering light quality. However, the impacts of fluorescent materials on aquatic plants and their environmental risks remain unclear. To assess the effects of luminescent materials on floating aquatic macrophytes and their rhizosphere microorganisms, 4-(di-p-tolylamino)benzaldehyde-A (DTB-A) and 4-(di-p-tolylamino)benzaldehyde-M (DTB-M) (emitting blue-green and orange-red light, respectively) were added individually and jointly to Spirodela polyrhiza cultures and set at different concentrations (1, 10, and 100 μM). Both DTB-A and DTB-M exhibited phytotoxicity, which increased with concentration under separate treatment. Moreover, the combined group exhibited obvious stress relief at 10 μM compared to the individually treated group. Fluorescence imaging showed that DTB-A and DTB-M were able to enter the cell matrix and organelles of plant leaves and roots. Peroxidation induced cellular damage, contributing to a decrease in superoxide dismutase (SOD) and peroxidase (POD) activities and malondialdehyde (MDA) accumulation. Decomposition of organelle structures, starch accumulation in chloroplasts, and plasmolysis were observed under the ultrastructure, disrupting photosynthetic pigment content and photosynthesis. DTB-A and DTB-M exposure resulted in growth inhibition, dry weight loss, and leaf yellowing in S. polyrhiza. A total of 3519 Operational Taxonomic Units (OTUs) were identified in the rhizosphere microbiome. The microbial communities were dominated by Alphaproteobacteria, Oxyphotobacteria, and Gammaproteobacteria, with the abundance and diversity varied significantly among treatment groups according to Shannon, Simpson, and Chao1 indices. This study revealed the stress defense response of S. polyrhiza to DTB-A and DTB-M exposures, which provides a broader perspective for the bioremediation of pollutants using aquatic plants and supports the further development of fluorescent materials for applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Key Message: Floral thermogenesis is an important reproductive strategy for attracting pollinators. We developed essential biological tools for studying floral thermogenesis using two species of thermogenic aroids, Symplocarpus renifolius and Alocasia odora. Aroids contain many species with intense heat-producing abilities in their inflorescences. Several genes have been proposed to be involved in thermogenesis of these species, but biological tools for gene functional analyses are lacking. In this study, we aimed to develop a protoplast-based transient expression (PTE) system for the study of thermogenic aroids. Initially, we focused on skunk cabbage (Symplocarpus renifolius) because of its ability to produce intense as well as durable heat. In this plant, leaf protoplasts were isolated from potted and shoot tip-cultured plants with high efficiency (ca. 1.0 × 10 5 /g fresh weight), and more than half of these protoplasts were successfully transfected. Using this PTE system, we determined the protein localization of three mitochondrial energy-dissipating proteins, SrAOX, SrUCPA, and SrNDA1, fused to green fluorescent protein (GFP). These three GFP-fused proteins were localized in MitoTracker-stained mitochondria in leaf protoplasts, although the green fluorescent particles in protoplasts expressing SrUCPA-GFP were significantly enlarged. Finally, to assess whether the PTE system established in the leaves of S. renifolius is applicable for floral tissues of thermogenic aroids, inflorescences of S. renifolius and another thermogenic aroid (Alocasia odora) were used. Although protoplasts were successfully isolated from several tissues of the inflorescences, PTE systems worked well only for the protoplasts isolated from the female parts (slightly thermogenic or nonthermogenic) of A. odora inflorescences. Our developed system has a potential to be widely used in inflorescences as well as leaves in thermogenic aroids and therefore may be a useful biological tool for investigating floral thermogenesis., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The study investigated the toxicity effects of 'form specific' engineered nanomaterials (ENMs) and ions released from nano-enabled products (NEPs), namely sunscreens, sanitisers, body creams and socks on Pseudokirchneriella subcapitata , Spirodela polyrhiza , and Daphnia magna . Additionally, risk estimation emanating from the exposures was undertaken. The ENMs and the ions released from the products both contributed to the effects to varying extents, with neither being a uniform principal toxicity agent across the exposures; however, the effects were either synergistic or antagonistic. D. magna and S. polyrhiza were the most sensitive and least sensitive test organisms, respectively. The most toxic effects were from ENMs and ions released from sanitisers and sunscreens, whereas body creams and sock counterparts caused negligible effects. The internalisation of the ENMs from the sunscreens could not be established; only adsorption on the biota was evident. It was established that ENMs and ions released from products pose no imminent risk to ecosystems; instead, small to significant adverse effects are expected in the worst-case exposure scenario. The study demonstrates that while ENMs from products may not be considered to pose an imminent risk, increasing nanotechnology commercialization may increase their environmental exposure and risk potential; therefore, priority exposure cases need to be examined.

Current knowledge on responses of aquatic clonal plants to resource availability is largely based on studies manipulating limited resource levels, which may have failed to capture the "big picture" for aquatic clonal plants in response to resource availability. In a greenhouse experiment, we grew the floating clonal plant Spirodela polyrhiza under ten nutrient levels (i.e., 1/64×, 1/32×, 1/16×, 1/8×, 1/4×, 1/2×, 1×, 2×, 4× and 8×full-strength Hoagland solution) and examined their responses in terms of clonal growth, morphology and biomass allocations. The responses of total biomass and number of ramets to nutrient availability were unimodal. A similar pattern was found for frond mass, frond length and frond width, even though area per frond and specific frond area fluctuated greatly in response to nutrient availability. In contrast, the responses of root mass and root length to nutrient availability were U-shaped. Moreover, S. polyrhiza invested more to roots under lower nutrient concentrations. These results suggest that nutrient availability may have distinct influences on roots and fronds of the aquatic clonal plant S. polyrhiza, resulting in a great influence on the whole S. polyrhiza population., Competing Interests: The authors have declared that no competing interests exist.

The plant circadian oscillation system is based on the circadian clock of individual cells. Circadian behavior of cells has been observed by monitoring the circadian reporter activity, such as bioluminescence of AtCCA1::LUC+. To deeply analyze different circadian behaviors in individual cells, we developed the dual-color bioluminescence monitoring system that automatically measured the luminescence of two luciferase reporters simultaneously at a single-cell level. We selected a yellow-green-emitting firefly luciferase (LUC+) and a red-emitting luciferase (PtRLUC) that is a mutant form of Brazilian click beetle ELUC. We used AtCCA1::LUC+ and CaMV35S::PtRLUC. CaMV35S::LUC+ was previously reported as a circadian reporter with a low-amplitude rhythm. These bioluminescent reporters were introduced into the cells of a duckweed, Lemna minor, by particle bombardment. Time series of the bioluminescence of individual cells in a frond were obtained using a dual-color bioluminescence monitoring system with a green-pass- and red-pass filter. Luminescence intensities from the LUC+ and PtRLUC of each cell were calculated from the filtered luminescence intensities. We succeeded in reconstructing the bioluminescence behaviors of AtCCA1::LUC+ and CaMV35S::PtRLUC in the same cells. Under prolonged constant light conditions, AtCCA1::LUC+ showed a robust circadian rhythm in individual cells in an asynchronous state in the frond, as previously reported. By contrast, CaMV35S::PtRLUC stochastically showed circadian rhythms in a synchronous state. These results strongly suggested the uncoupling of cellular behavior between these circadian reporters. This dual-color bioluminescence monitoring system is a powerful tool to analyze various stochastic phenomena accompanying large cell-to-cell variation in gene expression., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The study shows how microalgae biofilm formation and antioxidant responses to the production of reactive oxygen species (ROS) is alter by the presences of Lemna minor L., Chlorella vulgaris, and Aphanizomenon flos-aquae. The study involves the cultivation of the biofilm of Chlorella vulgaris and Aphanizomenon flos-aquae in three bioreactors. The condition of growth for the biofilm formation was varied across the three bioreactors to enable the dominance Chlorella vulgaris and Aphanizomenon flos-aquae in one of the bioreactors. Lemna minor L. was also introduce into one of the bioreactors to determine its effect on the biofilm formation. The result obtained shows that C. vulgaris and A. flos-aquae dominate the biofilm, resulting in a high level of H 2 O 2 and O 2 - (H 2 O 2 was 0.122 ± 0.052 and 0.183 ± 0.108 mmol/L in C. vulgaris and A. flos-aquae, respectively, and O 2 - was 0.261 ± 0.039 and 0.251 ± 0.148 mmol/L in C. vulgaris and A. flos-aquae, respectively). The study also revealed that the presence of L. minor L. tend to reduce the oxidative stress to the biofilm leading to low production of ROS (H 2 O 2 was 0.086 ± 0.027 and 0.089 ± 0.045 mmol/L in C. vulgaris and A. flos-aquae respectively, and O 2 - was 0.185 ± 0.044 and 0.161 ± 0.065 mmol/L in C. vulgaris and A. flos-aquae respectively). The variation in the ability of the biofilm of C. vulgaris and A. flos-aquae to respond via chlorophyll, carotenoid, flavonoid, anthocyanin, superoxide dismutase, peroxidase, catalase, glutathione reductase activities, antioxidant reducing power, phosphomolybdate activity, DPPH reduction activity, H 2 O 2 scavenging activity, lipid content and organic carbon also supports the fact that the presence of biomass of microalgae and aquatic macrophytes tend to affect the process of microalgae biofilm formation and the ability of the biofilm to produce antioxidant. This high nutrient utilization leads to the production of biomass which can be used for biofuel production and other biotechnological products., (Copyright © 2021. Published by Elsevier Inc.)

Watermeal, Wolffia australiana, is the smallest known flowering monocot and is rich in protein. Despite its great potential as a biotech crop, basic research on Wolffia is in its infancy. Here, we generated the reference genome of a species of watermeal, W. australiana, and identified the genome-wide features that may contribute to its atypical anatomy and physiology, including the absence of roots, adaxial stomata development, and anaerobic life as a turion. In addition, we found evidence of extensive genome rearrangements that may underpin the specialized aquatic lifestyle of watermeal. Analysis of the gene inventory of this intriguing species helps explain the distinct characteristics of W. australiana and its unique evolutionary trajectory., (© 2021. The Author(s).)

Successful eutrophication control strategies need to address the limiting nutrient. We conducted a battery of laboratory and in situ nutrient-limitation tests with waters collected from 9 streams in an agricultural region of the upper Snake River basin, Idaho, USA. Laboratory tests used the green alga Raphidocelis subcapitata, the macrophyte Lemna minor (duckweed) with native epiphytes, and in situ nutrient-limitation tests of periphyton were conducted with nutrient-diffusing substrates (NDS). In the duckweed/epiphyte test, P saturation occurred when concentrations reached about 100 μg/L. Chlorophyll a in epiphytic periphyton was stimulated at low P additions and by about 100 μg/L P, epiphytic periphyton chlorophyll a appeared to be P saturated. Both duckweed and epiphyte response patterns with total N were weaker but suggested a growth stimulation threshold for duckweed when total N concentrations exceeded about 300 μg/L and approached saturation at the highest N concentration tested, 1300 μg/L. Nutrient uptake by epiphytes and macrophytes removed up to 70 and 90% of the N and P, respectively. The green algae and the NDS nutrient-limitation test results were mostly congruent; N and P co-limitation was the most frequent result for both test series. Across all tests, when N:P molar ratios >30 (mass ratios >14), algae or macrophyte growth was P limited; N limitation was observed at N:P molar ratios up to 23 (mass ratios up to 10). A comparison of ambient periphyton chlorophyll a concentrations with chlorophyll a accrued on control artificial substrates in N-limited streams, suggests that total N concentrations associated with a periphyton chlorophyll a benchmark for desirable or undesirable conditions for recreation would be about 600 to 1000 μg/L total N, respectively. For P-limited streams, the corresponding benchmark concentrations were about 50 to 90 μg/L total P, respectively. Our approach of integrating controlled experiments and matched biomonitoring field surveys was cost effective and more informative than either approach alone., Competing Interests: The authors have declared that no competing interests exist.

Flavonoids may mediate UV protection in plants either by screening of harmful radiation or by minimizing the resulting oxidative stress. To help distinguish between these alternatives, more precise knowledge of flavonoid distribution is needed. We used confocal laser scanning microscopy (cLSM) with the "emission fingerprinting" feature to study the cellular and subcellular distribution of flavonoid glucosides in the giant duckweed (Spirodela polyrhiza), and investigated the fitness effects of these compounds under natural UV radiation and copper sulphate addition (oxidative stress) using common garden experiments indoors and outdoors. cLSM "emission fingerprinting" allowed us to individually visualize the major dihydroxylated B-ring-substituted flavonoids, luteolin 7-O-glucoside and luteolin 8-C-glucoside, in cross-sections of the photosynthetic organs. While luteolin 8-C-glucoside accumulated mostly in the vacuoles and chloroplasts of mesophyll cells, luteolin 7-O-glucoside was predominantly found in the vacuoles of epidermal cells. In congruence with its cellular distribution, the mesophyll-associated luteolin 8-C-glucoside increased plant fitness under copper sulphate addition but not under natural UV light treatment, whereas the epidermis-associated luteolin 7-O-glucoside tended to increase fitness under both stresses across chemically diverse genotypes. Taken together, we demonstrate that individual flavonoid glucosides have distinct cellular and subcellular locations and promote duckweed fitness under different abiotic stresses., (© 2020 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

The co-contamination of naphthalene (NAP) and microcystin-LR (MC-LR) commonly occurs in eutrophic waters. However, the joint effects of NAP and MC-LR on plants in aquatic environments remain unknown. Landoltia punctata is characterized by high starch yields and high biomass in polluted waters and has been proven to be a bioenergy crop and phytoremediation plant. In this study, L. punctata was cultured in a nutrient medium with environmentally relevant NAP (0.1, 1, 3, 5, and 10 μg/L) and MC-LR (5, 10, 25, 50, and 100 μg/L) to determine individual and joint toxic effects. The effects of NAP and MC-LR on physiological responses of L. punctata, including growth, starch accumulation, and antioxidant responses, were studied. Bioaccumulation of MC-LR in L. punctata, with or without NAP, was also examined. The results showed that growth and chlorophyll-a contents of L. punctata were reduced at high concentrations of MC-LR (≥ 25 μg/L), NAP (≥ 10 μg/L) and their mixture (≥ 10 + 1 μg/L) after exposure for 7 d. Starch accumulation in L. punctata did not decrease when exposed to NAP and MC-LR, and higher starch content of 29.8 % ± 2.7 % DW could be due to the destruction of starch-degrading enzymes. The antioxidant responses of L. punctata were stronger after exposure to MC-LR + NAP than when exposed to a single pollutant, although not enough to avoid oxidative damage. NAP enhanced the bioaccumulation of MC-LR in L. punctata when NAP concentration was higher than 5 μg/L, suggesting that higher potentials of MC-LR phytoremediation with L. punctata may be observed in NAP and MC-LR co-concomitant waters. This study provides theoretical support for the application of duckweed in eutrophic waters containing organic chemical pollutants., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Pharmaceuticals, which are designed to be biologically active at low concentrations, are found in surface waters, meaning aquatic organisms can be exposed to complex mixtures of pharmaceuticals. In this study, the adverse effects of four pharmaceuticals, 17α-ethynylestradiol (synthetic estrogen), methotrexate (anticancer drug), diclofenac (nonsteroidal anti-inflammatory drug) and fluoxetine (antidepressant), and their binary mixtures at mg/L concentrations were assessed using the 7-day Lemna minor test, with both apical and biochemical markers evaluated. The studied biochemical markers included chlorophyll a, chlorophyll b, carotenoids and oxidative stress enzymes catalase, glutathione-S-transferase and glutathione reductase, with effects compared to solvent controls. The adverse effects on Lemna minor were dose-dependent for frond number, surface area, relative chlorophyll content and activity of glutathione S-transferase for both individual pharmaceuticals and binary mixtures. According to the individual toxicity values, all tested pharmaceuticals can be considered as toxic or harmful to aquatic organisms, with methotrexate considered highly toxic. The most sensitive endpoints for the binary mixtures were photosynthetic pigments and frond surface area, with effects observed in the low mg/L concentration range. The concentration addition model and toxic unit approach gave similar mixture toxicity predictions, with binary mixtures of methotrexate and fluoxetine or methotrexate and 17α-ethynylestradiol exhibiting synergistic effects. In contrast, mixtures of diclofenac with fluoxetine, 17α-ethynylestradiol or methotrexate mostly showed additive effects. While low concentrations of methotrexate are expected in surface water, chronic ecotoxicological data for invertebrates and fish are lacking, but this is required to better assess the environmental risk of methotrexate., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Low temperature stress has a severe impact on the distribution, physiology, and survival of plants in their natural habitats. While numerous studies have focused on the physiological and molecular adjustments to low temperatures, this study provides evidence that cold induced physiological responses coincide with distinct ultrastructural alterations. Three plants from different evolutionary levels and habitats were investigated: The freshwater alga Micrasterias denticulata , the aquatic plant Lemna sp. , and the nival plant Ranunculus glacialis. Ultrastructural alterations during low temperature stress were determined by the employment of 2-D transmission electron microscopy and 3-D reconstructions from focused ion beam-scanning electron microscopic series. With decreasing temperatures, increasing numbers of organelle contacts and particularly the fusion of mitochondria to 3-dimensional networks were observed. We assume that the increase or at least maintenance of respiration during low temperature stress is likely to be based on these mitochondrial interconnections. Moreover, it is shown that autophagy and degeneration processes accompany freezing stress in Lemna and R. glacialis . This might be an essential mechanism to recycle damaged cytoplasmic constituents to maintain the cellular metabolism during freezing stress.

Duckweed is a simple aquatic floating plant having great potential in sewage treatment and bioenergy production. Duckweed rarely flowers in nature, which greatly limits its germplasm collection, conservation, and heterosis usage. Salicylic acid (SA) can efficiently induce flowering of duckweed (e.g., Lemna gibba); however, the related genes and regulatory networks remain unclear. In this work, we demonstrated that L. gibba flowering induced by SA was photoperiod-dependent, stress-involved, and abscisic acid (ABA)-disrupted. Totally 202, 78, and 413 differentially expressed (DE) genes were up-regulated, while 429, 72, and 307 were down-regulated at flower induction, flower initiation, and flowering stages, respectively. At the flower induction stage, the down-regulated genes were mainly involved in cell wall, auxin and ABA, light reaction, and abiotic stress, while the up-regulated genes were involved in development, brassinosteroid, major CHO metabolism, and redox. At the flower initiation stage, the down-regulated genes were enriched in light reaction and lipid metabolism, whereas the up-regulated genes were enriched in starch degradation and Ca 2+ signaling. At the flowering stage, the down-regulated genes were significantly enriched in photosynthesis, gibberellic acid, starch synthesis, nitrogen metabolism, and redox, while the up-regulated genes were enriched in cell wall, jasmonic acid, secondary metabolism, and Ca 2+ signaling. Besides, 46 transcription factors and 13 flowering-related DE genes were identified. Finally, a possible floral pathway, where LgTEM1, LgSVP, and LgFT1 might play critical roles in SA-induced flowering in L. gibba, was discussed. These findings provide a useful foundation for further investigation of genes and regulatory networks of SA-induced flowering in duckweed., (Copyright © 2020. Published by Elsevier Masson SAS.)

In the literature, there is a lack of data on the effect of gentian violet (GV) and congo red (CR) dyes on the biosynthesis pathway of biogenic amines (BAs) in Lemna minor L. (common duckweed). This plant species is an important link in the food chain. Both dyes inhibited growth, biomass yield and the biosynthesis of chlorophyll a in common duckweed. The predicted toxic units demonstrated that GV had a more toxic effect on the growth rate and biomass yield of common duckweed than CR. Decarboxylase activity in the biosynthesis of BAs in common duckweed is also a useful indicator for evaluating the toxicity of both dyes. Gentian violet also exerted more phytotoxic effects on the analyzed biochemical features of common duckweed because it changed the putrescine (Put) biosynthesis pathway, increased tyramine content 1.6 fold, inhibited the activity of S-adenosylmethionine decarboxylase by 40% and the activity of ornithine decarboxylase (ODC) by 80%. Tyrosine decarboxylase (TDC) was most active in plants exposed to the highest concentration of GV. Similarly to control plants, in common duckweed exposed to CR, Put was synthesized from ornithine; however, spermidine content was 86% higher, Put content was 51% lower, and ODC activity was 86% lower., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Plant diversity has important functions in ecosystem productivity overyielding and community stability. Little is known about the mechanism causing productivity overyielding and stability under harsh conditions. This study investigated the photosynthetic response and subcellular distribution of uni- and co-cultured duckweeds (Lemna aequinoctialis and Spirodela polyrhiza) under excess copper (1.0 mg/L) and low temperature (5 °C) conditions. The results showed that the growth of uni-cultured L. aequinoctialis was not different from that of uni-cultured S. polyrhiza across copper treatments at control temperature (25 °C). The growth rate of L. aequinoctialis increased by 55.5 % under excess copper concentration when it coexisted with S. polyrhiza, compared with uni-culture. Subcellular distributions of copper were predominantly distributed in cell walls. S. polyrhiza accumulated more copper in cell walls than L. aequinoctialis under uni-cultured condintion at excess copper concentration. Co-cultured S. polyrhiza increased copper accumulation in cell walls of co-cultured L. aequinoctialis to decrease toxicity at excess copper concentration, compared with L. aequinoctialis. Low temperature increased copper toxicity, with duckweeds having lower growth rate and photosynthetic activities (Fv/Fm). The L. aequinoctialis growth rate in co-culture was higher than in uni-culture under excess copper concentration and low temperature conditions, indicating that S. polyrhiza decreased the copper toxicity for L. aequinoctialis. The photosynthetic activity (Fv/Fm) of co-cultured L. aequinoctialis was higher than that of uni-cultured L. aequinoctialis exposed to excess copper concentration at low temperature. The community that formed by co-culturing S. polyrhiza and L. aequinoctialis produced more biomass by avoiding the toxicity of excess copper through heavy metal compartmentalization and photosynthetic activities., Competing Interests: Declaration of Competing Interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In the present study, single and mixture effects of cadmium (Cd) and zinc (Zn) on Lemna gibba were analyzed and compared using growth parameters, based on frond number and fresh weight, and biochemical parameters, such as pigment, protein content and activity of antioxidant enzymes. Plants were exposed for 7 days to these metals in nutrient solution. Single and mixture exposures affected plant growth and the biomarkers of the antioxidant response. Considering the growth parameters, Cd was found to be much more toxic than Zn. IC50-7d, based on growth rate calculated on frond number, were 17.8 and 76.73 mg/L, and on fresh weight were 1.08 and 76.93 mg/L, for Cd and Zn respectively. For Cd, LOEC values were obtained at 2.06 and 1.03 mg/L, for frond number and fresh weight respectively; while for Zn, at 20.1 and 74.6 mg/L. A high toxicity effect, considering the same response variables, was observed in plants exposed to the mixtures. Three fixed ratios, based on toxic units (TU) were assayed, ratio 1: 2/3 Cd-1/3 Zn, ratio 2: 1/2 Cd-1/2 Zn and ratio 3: 1/3 Cd-2/3 Zn. Ratio 3 (where Zn was added in higher proportion) was the less toxic. All concentrations of Ratio 1 and 2 significantly inhibited plant growth, showing a 100% inhibition of growth rate at the highest concentrations when based on frond number. Catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APOX; EC 1.11.1.11) and guaiacol peroxidase (GPOX; EC 1.11.1.7) activities in single metals assays were higher than controls. In mixture tests, the activity of APOX and GPOX was significantly stimulated in plants exposed to all evaluated combinations, while CAT was mainly stimulated in Ratio 3. It was observed that the activity of the enzymes was increased in the mixtures compared with similar concentrations evaluated individually. APOX activity was observed to fit the CA model and following a concentration-response pattern. The response of this antioxidant enzyme could serve as a sensitive stressor biomarker for Cd-Zn interactions. Frond number in Cd-Zn mixtures was not well predicted from dissolved metal concentration in solution using concentration addition (CA) as reference model, as results showed that toxicity was more than additive, with an average of ΣTU = 0.75. This synergistic effect was observed up to 50 mg Zn/L in the mixture, but when it was present in higher concentrations a less than additive effect was observed, indicating a protective effect of Zn. A synergistic and dose-ratio deviations from CA model were also observed.

Vertical farming using light-emitting diode offers potential for the early production phase (few weeks) of young ornamental plants. However, once transferred to the greenhouse, the photosynthetic acclimation of these young plants might depend on this initial light regime. To obtain insight about this acclimatization, Chrysanthemum (sun species) and Spathiphyllum (shade species) were preconditioned in growth chambers for 4 weeks under four light qualities: blue (B), red (R), red/blue (RB, 60% R) and white (W) at 100 μmol m -2 s -1 . Monochromatic light (R and B) limited leaf development of both species, which resulted in a lower leaf mass per area when compared to multispectral light (RB for Chrysanthemum, RB and W for Spathiphyllum). R-developed leaves had a lower photosynthetic efficiency in both species. After the light quality pretreatment, plants were transferred to the greenhouse with high-intensity natural light conditions. On the first day of transfer, R and B preconditioned leaves of both species had an inhibited photosynthesis. After 1 week in natural light condition, rapid light curve parameters of Chrysanthemum leaves that developed under B acclimated to sunlight had a similar level than RB-developed leaves unlike R-leaves. Spathiphyllum leaves showed a decrease in maximum electron transport rate and this was most pronounced for the R pretreatment. After 1 month, R-preconditioned Chrysanthemum had the lowest dry mass, while no effects on the dry weight of Spathiphyllum with respect to the pretreatments were observed. Light quality during preconditioning affected the leaf ability to acclimate to natural high light intensities in greenhouse environment., (© 2020 Scandinavian Plant Physiology Society.)

Most theory on the evolution of senescence implicitly assumes that all offspring are of equal quality. However, in addition to age-related declines in survival and fecundity (classically defined senescence), many organisms exhibit age-related declines in offspring quality, a phenomenon known as a parental age effect. Theoretical work suggests that parental age effects may alter age trajectories of selection and therefore shape the evolution of senescence; however, to date, these analyses have been limited to idealized life cycles and models of maternal care in human populations. To gain a broader understanding of how parental age effects may shape age trajectories of selection, we extend the classic age-structured population projection model to also account for parental age structure and apply this model to empirical data from an aquatic plant known to exhibit parental age effects (the duckweed Lemna minor ), as well as a diverse set of simulated life cycles. Our results suggest that parental age effects alter predictions from classic theory on the evolution of senescence. Age-related declines in offspring quality reduce the relative value of late-life reproduction, leading to steeper age-related declines in the force of natural selection than would otherwise be expected and potentially favoring the evolution of more rapid rates of senescence.

In order to evaluate the differential absorption and toxicity of arsenate (As V ) and arsenite (As III ), Lemna valdiviana plants were grown in a nutrient solution and subjected to 0.0 (control); 0.5; 1.0; 1.5; 2.0; 3.0; 4.0; 5.0 and 7.5 mg L -1 of As III or As V for three days. Exposure to both chemical forms resulted in As bioaccumulation, although As III -grown plants showed higher As content in tissues. In As V -grown plants, the relative growth rate (RGR) decreased to 50%, at a concentration of 4.0 mg L -1 , while for treatments with As III , the same decrease was observed at 1.0 mg L -1 . The tolerance index decreased with increasing concentrations, with lower values for As III . Plants treated with As III showed increased superoxide anion levels, whilst higher levels of hydrogen peroxide were present in AsV-treated plants. Moreover, malondialdehyde (MDA) levels were higher for plants subjected to As III when compared to As V at lower concentrations. Concentrations of 1 mg L -1 of As III and 4 mg L -1 of As V showed equivalent MDA levels. Superoxide dismutase and catalase activities were increased at low concentrations and were inhibited at higher concentrations of As III and As V , whereas peroxidase activity was positively modulated by increased As III or As V concentrations. In conclusion, L. valdiviana plants took up and accumulated arsenic as As III or As V , demonstrating the potential for phytoremediation of this metalloid. Furthermore, As III -exposed plants showed enhanced toxicity when compared to As V , at the same applied concentration, although toxicity was more related to internal As concentrations, regardless of the chemical form applied.

By the year 2100, temperatures are predicted to increase by about 6 °C at higher latitudes and about 3 °C in the tropics. In spite of the smaller increase in the tropics, consequences may be more severe because the climatic niches of tropical species are generally assumed to be rather narrow due to a high degree of climate stability and higher niche specialisation. However, rigorous data to back up this notion are rare. We chose the megadiverse genus Anthurium (Araceae) for study. Considering that the regeneration niche of a species is crucial for overall niche breadth, we focused on the response of germination and early growth through a temperature range of 24 °C of 15 Anthurium species, and compared the thermal niche breadth (TNB) with the temperature conditions in their current range, modelled from occurrence records. Surprisingly, an increase of 3 °C would lead to a larger overlap of TNB of germination and modelled in situ temperature conditions, while the overlap of TNB of growth with in situ conditions under current and future conditions is statistically indistinguishable. We conclude that future temperatures tend to be closer to the thermal optima of most species. Whether this really leads to an increase in performance depends on other abiotic and biotic factors, most prominently potentially changing precipitation patterns., (© 2019 The Authors. Plant Biology published by John Wiley & Sons Ltd on behalf of German Society for Plant Sciences, Royal Botanical Society of the Netherlands.)

This study involved the development of mathematical linear regression models to describe the relationships between mean plant biomass (M) and population density (D), M and frond diameter (L), frond numbers (N) and L of Lemna minor under different initial population densities (3200, 4450, and 6400 plants/m 2 ), respectively, from the perspective of the self-thinning law. Our results revealed that the value of the allometric exponents for M and D were - 3/2. Further, the concentrations of Zn, Pb, Cu, Fe, and Ni accumulated in L. minor plants were 0.86, 0.32, 0.36, 0.62, and 0.39 mg/kg, respectively. Based on these developed equations and the heavy metal accumulations by L. minor, the phytoremediation capacity of L. minor was quantified via its frond diameters. Overall, the present study provides a cost-effective green method for managing the phytoremediation of heavy metal-contaminated aquatic environments.

A complete understanding of the plant microbiome has not yet been achieved due to its complexity and temporal shifts in the community structure. To overcome these issues, we created a synthetic bacterial community of the aquatic plant, duckweed. The synthetic community established with six bacterial strains showed a stable composition for 50 days, which may have been because duckweed maintains a similar physiological status through its clonal reproduction. Additionally, the synthetic community reflected the taxonomic structure of the natural duckweed microbiome at the family level. These results suggest the potential of a duckweed-based synthetic community as a useful model system for examining the community assembly mechanisms of the plant microbiome.

The bioluminescent reporter system is a powerful tool for the long-term monitoring of gene expression because of its noninvasive nature. Furthermore, in combination with high-sensitive imaging technology, spatiotemporal analysis on regulation and heterogeneity in gene expression is possible. We developed a single-cell bioluminescent imaging system for plants through a transient gene transfection by particle bombardment. By applying this system to a duckweed species, we succeeded in monitoring circadian rhythms of individual cells in an intact plant for over a week. Here we describe methods for gene transfection by particle bombardment and single-cell bioluminescence monitoring by a high-sensitive camera. This technique provides a platform for characterizing gene expression patterns of individual cells in the same tissue.

Management of leachates generated by solid waste disposal is a very challenging aspect of landfill management in most parts of the world. In most developing countries, the leachates generated are discharged into the environment without treatment, leading to contamination of ground and surface waters and causing human health problems. Even though its potential risk has been established through chemical analyses, less work has been conducted on its effect on ecosystems. This study assessed the toxicity of leachates from three landfill sites of different ages from Ghana, namely Tema, Mallam and Oblogo, to aquatic organisms. Duckweed (Lemna minor) and crustaceans (Daphnia magna) toxicity tests were performed using exposures to concentrations of 6.25, 12.5, 25, 50 and 100 mL/L of the landfill leachates in control growth media. Physico-chemical properties of the leachates were also determined. The leachates from all the sites were toxic with IC 50 values ranging from 2.8 to 29.5%. The Oblogo landfill leachate (the oldest site) being most toxic to duckweed and Tema landfill leachate (the youngest site) most toxic to D. magna. Leachates characterized had varying concentrations of heavy metals (0.2-42.3 mg/L) with Cu and Cd below detectable limit. The organic component COD was below the permissible level (110-541 mg/L) and the TOC exceeded the permissible level (350-6920 mg/L). These results indicate that the age and other characteristics of the landfill sites contribute to the difference in the toxicity of the Ghana landfill leachates., (Copyright © 2019 Elsevier B.V. All rights reserved.)

There is increasing concern about the effects of releasing emerging contaminants (i.e. endocrine-disrupting chemicals, pharmaceuticals, personal-care products and flame retardants) into the environment. Particular attention is being paid to perfluoroalkyl substances (PFAS) because of their persistence and bioaccumulation, especially in the aquatic environment. In this paper, we present results of a study aimed at evaluating the effects of different perfluorooctanoic acid (PFOA) concentrations (2, 20 and 200 μg/L) on morpho-physiological traits in Lemna minor L. plants. The accumulation of PFOA in the plant's tissues was also monitored. L. minor was selected as a model plant for ecotoxicological studies, and we performed a seven-day assay for this investigation. The results highlight the lack of inhibitory effects on biometric parameters such as mean frond area, total frond number, multiplication rate, doubling time of frond number and average specific growth rate, for each of tested PFOA concentrations. Also, at photosynthetic level, physiological measurements showed that chlorophyll content and electron transport rate (ETR) were not affected by the exposure to PFOA. Remarkably, the chlorophyll fluorescence images, used for the first time in a study on PFOA, evidenced no impairment to the photosynthetic efficiency, measured by the maximum quantum yield of photosystem II (PSII) photochemistry (Fv/Fm), the quantum efficiency of PSII photochemistry (ΦPSII) and the non-photochemical quenching (NPQ) over the leaf surface of PFOA-treated plants, in comparison to control. Quantification of PFOA in the growth medium at the end of the seven-day test revealed no statistically different concentrations in plates with or without L. minor plants. We detected increasing PFOA accumulation in plant tissues, in accordance with the PFOA concentrations in the medium. Therefore, the L. minor plants were capable of taking up and accumulating PFOA. The ecological impact of the environmentally relevant PFOA concentrations tested in this work on biological organisms of the aquatic environment is discussed., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background & Aims: While the world is extensively looking for alternatives to animal protein sources, it is not clear which plant sources can provide the requisite full complement of essential amino acids (EAAs). Wolffia globosa is an aquatic, edible duckweed, the smallest plant on earth, and it offers all nine EAAs, dietary fibers, polyphenols, iron, zinc and B 12 vitamin. This work was designed to evaluate Mankai (a newly developed high-protein strain of W. globosa) as an optional bioavailable source of EAAs for humans (primary outcome), and of further nutrients such as vitamin B 12 , in comparison to well-established animal and plant protein sources; cheese and peas, respectively., Methods: 36 men, subjected for 3 days to a stable diet and subsequent overnight (12 h) fast, were randomized to consume one of three iso-protein (30 g) based test-meals (soft cheese, green peas, Mankai). Blood samples were collected at 0, 30, 90 and 180 min., Results: The 3 h blood concentrations of the EAAs: histidine, phenylalanine, threonine, lysine, and tryptophan, triggered by intake of Mankai, was essentially significant as compared to baseline (p < 0.05) and similar to that of soft cheese and pea changes (p > 0.05 between groups). Although branched-chain-amino-acids (leucine/isoleucine, valine) increased significantly by Mankai within 3 h (p < 0.05 vs. baseline), the change was relatively higher for cheese as compared to Mankai or peas (p < 0.05 between groups). The increase in vitamin B 12 by Mankai was higher as compared to changes induced by either cheese (p=0.007) or peas (p=0.047, between groups)., Conclusions: Mankai may provide a high-quality substitute source for animal protein, and a potential bioavailable source of vitamin B 12 ., (Copyright © 2018 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Clonal propagation allows some plant species to achieve massive population sizes quickly but also reduces the evolutionary independence of different sites in the genome. We examine genome-wide genetic diversity in Spirodela polyrhiza, a duckweed that reproduces primarily asexually. We find that this geographically widespread and numerically abundant species has very low levels of genetic diversity. Diversity at nonsynonymous sites relative to synonymous sites is high, suggesting that purifying selection is weak. A potential explanation for this observation is that a very low frequency of sex renders selection ineffective. However, there is a pronounced decay in linkage disequilibrium over 40 kb, suggesting that though sex may be rare at the individual level it is not too infrequent at the population level. In addition, neutral diversity is affected by the physical proximity of selected sites, which would be unexpected if sex was exceedingly rare at the population level. The amount of genetic mixing as assessed by the decay in linkage disequilibrium is not dissimilar from selfing species such as Arabidopsis thaliana, yet selection appears to be much less effective in duckweed. We discuss alternative explanations for the signature of weak purifying selection., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Cadmium (Cd) is a serious threat to plants health. Though some genes have been reported to get involved in the regulation of tolerance to Cd, the mechanisms underlying this process are not fully understood. Na + /H + antiporter (NHX1) plays an important role in Na + /H + trafficking. The salt and cadmium stress tolerance were found to be enhanced by NHX1 in duckweed according to our previous study, however, its function in Cd 2+ flux under Cd stress has not been studied. Here we explored the Cd 2+ flux in wild type (WT) and NHX1 transgenic duckweed (NHX1) under Cd stress. We found that the Cd 2+ influx in NHX1 duckweed was significantly declined, followed by an increased Cd 2+ efflux after 20 min treatment of Cd, which resulted a less accumulation of Cd in NHX1. Reversely, inhibition of NHX1 by amiloride treatment, enhanced Cd 2+ influx in NHX1 duckweed, subsequently delayed Cd 2+ efflux in both genotypes of duckweed under Cd 2+ shock. H + efflux in NHX1 duckweed was lower compare with that in WT with 20 min Cd 2+ shock. NHX1 also increased the pH value with Cd 2+ stress in the transgenic rhizoid. These finding suggested a new function of NHX1 in regulation of Cd 2+ and H + flow during short-term Cd 2+ shock., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The global occurrence of pharmaceuticals in the aquatic environment has been considered a particularly concerning problem with unknown consequences. Non-steroidal anti-inflammatory drugs (NSAIDs) including diclofenac (DCF) and salicylic acid (SA), are among the most frequently prescribed drugs in the world, being consequently commonly found in the aquatic environment. Prolonged experiments (with duration of exposure that surpass those recommended by already established testing guidelines) are important to obtain ecologically relevant data to address the issue of NSAIDs ecotoxicity, because by being more realistically (namely in terms of levels and durations of exposure), such tests may indicate realistic challenges posed to aquatic organisms. Among the most common test species that are used for assessing environmental quality, plants play a leading role. Lemna species are among the most important plants used for ecotoxicity testing. Therefore, the aim of this study was to evaluate the temporal effect of a prolonged exposure of DCF and SA on Lemna minor. To attain this purpose, L. minor plants were chronically exposed to 0, 4, 20, and 100 μg/L of both pharmaceuticals, and samplings were performed at 6, 10 and 14 days of exposure. The analyzed endpoints were: levels of chlorophyll a, b and total, carotenoids; and enzymatic biomarkers, such as catalase, ascorbate peroxidase and glutathione-S-transferases. Diclofenac was responsible for alterations in all analyzed parameters in different intervals of exposure. Salicylic acid exposure was not capable of causing alterations on pigment contents of L. minor, however, enzymatic biomarkers were altered at all sampling intervals. Thus, it is possible to conclude that both pharmaceuticals can cause damage on the tested macrophyte species, biochemical parameters being more sensitive than physiological ones. Additional prolonged experiments are required to understand the chronic effects of different pharmaceuticals in the aquatic environment, especially in plants., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Boron (B) in the irrigation water can be hazardous to human beings and other aquatic or terrestrial organisms when B concentration exceeds a certain level. More importantly, B removal from irrigation water is relatively difficult using conventional processes. In the present experiment, an innovative treatment model based on monoculture and polyculture duckweed wastewater treatment modules was tested for B-rich irrigation water purification and bioelectricity harvesting. Different modules were designed using Lemna gibba L., Lemna minor L., and their combination in order to determine the most optimal duckweed species and vegetation structure for B removal process and bioelectricity generation in a module. In this respect, the module with a monoculture of Lemna gibba achieved the highest net B removal efficiency (71%) when it was exposed to 4 mg/L B (initial concentration). However, B removal efficiencies from all modules decreased when the initial B concentrations reached up to 4 mg/L in the irrigation water. The highest bioelectricity production was measured as 1.04 V with 17783 mWatt/m 2 power density at a current density of 44.06 mA/m 2 for module with Lemna gibba in monoculture through sacrificial magnesium anode. Specifically, both monocultures and polyculture removed considerable amounts of organic matter from irrigation water. However, biomass production and total chlorophyll (a + b) concentrations of duckweeds significantly decreased when they were exposed to 32 mg/L B in the irrigation water samples. Consequently, our modules present a holistic perspective to the prevention B toxicity problems in agricultural zones, and are a sustainable strategy for farmers or agricultural experts to produce bioelectricity by a cost-effective and eco-technological method.

Aquatic plants have to adapt to the environments distinct from where land plants grow. A critical aspect of adaptation is the dynamics of sequence repeats, not resolved in older sequencing platforms due to incomplete and fragmented genome assemblies from short reads. Therefore, we used PacBio long-read sequencing of the Spirodela polyrhiza genome, reaching a 44-fold increase of contiguity with an N50 (a median of contig lengths) of 831 kb and filling 95.4% of gaps left from the previous version. Reconstruction of repeat regions indicates that sequentially nested long terminal repeat (LTR) retrotranspositions occur early in monocot evolution, featured with both prokaryote-like gene-rich regions and eukaryotic repeat islands. Protein-coding genes are reduced to 18,708 gene models supported by 492,435 high-quality full-length PacBio complementary DNA (cDNA) sequences. Different from land plants, the primitive architecture of Spirodela 's adventitious roots and lack of lateral roots and root hairs are consistent with dispensable functions of nutrient absorption. Disease-resistant genes encoding antimicrobial peptides and dirigent proteins are expanded by tandem duplications. Remarkably, disease-resistant genes are not only amplified, but also highly expressed, consistent with low levels of 24-nucleotide (nt) small interfering RNA (siRNA) that silence the immune system of land plants, thereby protecting Spirodela against a wide spectrum of pathogens and pests. The long-read sequence information not only sheds light on plant evolution and adaptation to the environment, but also facilitates applications in bioenergy and phytoremediation., Competing Interests: The authors declare no conflict of interest.

Phenylurea herbicides are often present in the aquatic ecosystems and may be accumulated by the non-targeted organisms and impose a negative effect on the organism and the community. This study aims to investigate and compare the effects of two different isoproturon (IPU) pulse exposure scenarios on the non-targeted aquatic plant Lemna minor with effects observed in the standard test with continuous exposure. The obtained results showed that continuous IPU treatment causes significant reduction of photosynthetic pigment concentration and proteins as well as inhibition of L. minor growth. The activities of CAT, G-POX, and APX were significantly induced to diminish the accumulation of ROS under IPU treatment, but the induction of antioxidant enzymes was not sufficient to protect the plants from herbicide-induced oxidative stress. The growth of L. minor under pulse exposure to IPU recovers fast, but pulse treatment results in significant physiological changes in treated plants. The accumulation of H 2 O 2 and lipid peroxidation products, alongside the reduced concentration of proteins and photosynthetic pigments in pulse treatment after a recovery period, indicates that IPU causes prolonged oxidative stress in L. minor plants. The recovery potential of L. minor plants after treatment with herbicides may have an important role in maintaining the population of essential primary producers in aquatic ecosystems, but IPU-induced physiological changes could potentially have a significant role in modulating the response of the plants to the next exposure event., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The aim of this study was to compare the ecotoxicity of nickel (Ni) and zinc (Zn) assayed as single and as binary mixture. In addition, how were affected the population growth rates and oxidative stress biomarkers, comparing single to binary exposures. The toxicity tests were performed on Lemna gibba using a 7-day test. All calculations were made using measured total dissolved metal concentrations. IC50-7d, based on growth rate calculated on frond number and fresh weight, were 2.47/3.89 mg/L, and 76.73/76.93 mg/L, for Ni and Zn, respectively. Single metals affected plant growth following a non-linear concentration-response relationship. LOEC values for each metal were obtained at 0.92 and 20.1 mg/L for Ni and Zn, respectively. Biomarkers of the antioxidant response like Catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APOX; EC 1.11.1.11) and guaiacol peroxidase (GPOX; EC 1.11.1.7) activities in single metals assays were higher than controls, but when similar concentrations were added as mixtures, that increase was reduced and inhibition with respect to the control was observed for GPOX. APOX showed the highest activity. The concentration addition (CA) approach was evaluated and resulted in a correct predictor of Ni-Zn mixture toxicity on Lemna gibba. This was made comparing the EC50 and LOEC, measured taking the growth rate as endpoint, with those expected values according to the CA model. However, the measured biomarkers indicating a positive response to free radicals did not fit to concentration addition model when assayed in the binary mixture. Also, the main activity response of these was observed within a range of concentrations below the LOEC values for the mixture.

The Pistia stratiotes L. was tested for phytoremediation potential of the compound clomazone in water. Clomazone is a post-emergent herbicide marketed as Gamit ® . Five groups with four samples each were evaluated, a low concentration control (LCC: 37.86 mg L -1 ), low concentration treatment (LCT: 38.16 mg L -1 ), high concentration control (HCC: 54.71 mg L -1 ), high concentration treatment (HCT: 54.33 mg L -1 ), and a plant control group (PCG). Plant resistance to clomazone at determined concentrations and their ability to remove the herbicide from water by HPLC over 24 days were evaluated. The results demonstrate that P. stratiotes has high resistance to clomazone exposure and was able to eliminate up to 90% of the herbicide residues during the experimental period. Under dissipation by P. stratiotes in water, clomazone had a halflife of 19.6 days for in the control treatments, LCC and HCC, and 8.0 days in the treatment groups, LCT and HCT. This study indicates that Pistia stratiotes is an effective phytoremediation agent for the herbicide clomazone in water., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The Lemnaceae family comprises aquatic plants of angiosperms gaining attention due to their utility in wastewater treatment, and rapid production of biomass that can be used as feed, fuel, or food. Moreover, it can serve as a model species for neotenous growth and environmental adaptation. The latter properties are subject to post-transcriptional regulation of gene expression, meriting investigation of how miRNAs in Spirodela polyrhiza, the most basal and most thoroughly sequenced member of the family, are expressed under different growth conditions. To further scientific understanding of its capacity to adapt to environmental cues, we measured miRNA expression and processing of their target sequences under different temperatures, and in the presence of abscisic acid, copper, kinetin, nitrate, and sucrose. Using two small RNA sequencing experiments and one degradome sequencing experiment, we provide evidence for 108 miRNAs. Sequencing cleaved mRNAs validated 42 conserved miRNAs with 83 targets and 24 novel miRNAs regulating 66 targets and created a list of 575 predicted and verified targets. These analyses revealed condition-induced changes in miRNA expression and cleavage activity, and resulted in the addition of stringently reviewed miRNAs to miRBase. This combination of small RNA and degradome sequencing provided not only high confidence predictions of conserved and novel miRNAs and targets, but also a view of the post-transcriptional regulation of adaptations. A unique aspect is the role of miR156 and miR172 expression and activity in its clonal propagation and neoteny. Additionally, low levels of 24 nt sRNAs were observed, despite the lack of recent retrotransposition., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Environmentally mediated sensitivity of Lemna minor to copper (Cu) was evaluated for the first time in three experiments: the effects of two levels of nutrient concentration, light irradiance or Cu pre-exposure were tested. Various Cu concentrations (ranging from 0.05 to 0.25 mg/L) were used to assess the sensitivity of L. minor to this metal, using one common strain previously acclimatized to two different levels of light intensity, nutrient enrichment and Cu pre-exposure. Our results showed a phenotypic plastic response of the relative growth rates based on frond number and fresh mass production, and maximum quantum yield of photosystem II (F v /F m ). Growth was affected by the three environmental conditions both prior and during Cu exposure, whereas F v /F m was mostly affected during Cu exposure. Copper significantly influenced all the parameters measured in the three experiments. Environmental conditions significantly modified L. minor sensitivity to Cu in all experiments, with up to twofold difference depending on the treatment. Growth rate was the parameter that was most impacted. Our study revealed for the first time the existence of phenotypic plasticity in L. minor sensitivity to chemical contamination, and implies that environmental context needs to be taken into account for a relevant risk assessment.

Mycotoxins are an important class of chemicals of emerging concern, recently detected in aquatic environments, potentially reflecting the influence of fungicide resistance and climatic factors on fungal diseases in agricultural crops. Zearalenone (ZON) is a mycotoxin formed by Fusarium spp. and is known for its biological activity in animal tissues; both in vitro and in vivo. ZON has been reported in US and Polish surface waters at 0.7 - 96 ng/L, with agricultural run-off and wastewater treatment plants being the likely sources of mycotoxins. As some mycotoxins can induce phytotoxicity, laboratory studies were conducted to evaluate the toxicity of ZON (as measured concentrations) to freshwater algae (Pseudokirchneriella subcapitata) and macrophytes (Lemna minor) following OECD test guidelines 201 and 221, respectively. Zinc sulphate was used as a positive control. In the OECD 201 algal static study (72 h at 24 ± 1 °C), exposure to ZON gave average specific growth rate (cell density) EC 50 and yield (cell density) EC 50 values of > 3.1 and 0.92 (0.74 - 1.8) mg/L, respectively. ZON was less toxic in the OECD 221 static study and after 7 d at 24 ± 1 °C. L. minor growth was significantly reduced based on frond number and frond area at 11.4 mg ZON/L, showing a higher tolerance than reported for other mycotoxins with Lemna spp. Chlorophyll fluorescence parameters were used as biomarkers of impacts on photosystem II efficiency, with no effect seen in algae but, with responses being observed in L. minor between 5.2 - 14.4 mg ZON/L. ZON toxicity seen here is not of immediate concern in context with environmental levels, but this study highlights that other freshwater organisms including algae are more sensitive to mycotoxins than Lemna sp., the only current source of toxicity data for freshwater plants., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Caladium bicolor is an excellent landscape and pot plant grown for its ornamental value due to the varied shapes and multi-colored foliage. However, the ornamental value of in vitro clonally propagated plants is affected by the occurrence of morpho-physiological anomalies in the long-term cultures. During the long-term culture of rhizome-derived shoot cultures of C. bicolor cv. "Bleeding hearts", some incidences of hyperhydricity and albinism were observed. Hyperhydricity occurred at high frequency in the shoot cultures and was more prevalent in the liquid over solid medium cultivated plantlets. The addition of silver nitrate at 7.5 μM reduced prevalence of the condition over other treatment concentrations but, changes in the gelling agent concentration and photoperiodic incubation were ineffective. Albinism also occurred at a higher frequency but, was more prevalent in the cultures incubated below or above 12 h light/dark photoperiods. Evaluation of the physiological, biochemical parameters showed differences between the leaves of albino, hyperhydric and normal green C. bicolor micro shoots. The experimental results obtained suggested an effective role of pigment molecules, cellular osmolytes, and enzymatic and non-enzymatic antioxidant systems in the development of albino and hyperhydric leaves in C. bicolor micro shoot cultures. Photoperiodic incubation and culture conditions showed some effect on the incidences of hyperhydricity and albinism in the shoot cultures., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This paper aims to evaluate the effects of a product containing colloidal silver in the aquatic environment, using duckweed Lemna gibba as a model plant. Therefore, growth parameters, photosynthetic pigments content and protein content as physiological indices were evaluated. Changes in the content of non-enzymatic antioxidants and activity of several antioxidant enzymes, alongside with the accumulation of hydrogen peroxide and lipid peroxidation end-products were assessed to explore the potential of colloidal silver to induce oxidative stress. The commercially available colloidal silver product contained a primary soluble form of silver. The treatment with colloidal silver resulted in significant physiological and biochemical changes in L. gibba plants and a consequent reduction of growth. Accumulation of silver caused altered nutrient balance in the plants as well as a significant decrease in photosynthetic pigments content and protein concentration. The antioxidative response of L. gibba plants to treatment with colloidal silver was inadequate to protect the plants from oxidative stress caused by metal accumulation. Silver caused concentration-dependent and time-dependent hydrogen peroxide accumulation as well as the elevation of lipid peroxidation levels in L. gibba plants. The use of commercially available products containing colloidal silver, and consequent accumulation of silver, both ionic and nanoparticle form in the environment, represents a potential source of toxicity to primary producers in the aquatic ecosystem., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Thermogenesis in plants involves significant increases in their cyanide-resistant mitochondrial alternative oxidase (AOX) capacity. Because AOX is a non-proton-motive ubiquinol oxidase, the dramatic drop in free energy between ubiquinol and oxygen is dissipated as heat. In the thermogenic skunk cabbage (Symplocarpus renifolius), SrAOX is specifically expressed in the florets. Although SrAOX harbours conserved cysteine residues, the details of the mechanisms underlying its redox regulation are poorly understood. In our present study, the two mitochondrial thioredoxin o cDNAs SrTrxo1 and SrTrxo2, were isolated from the thermogenic florets of S. renifolius. The deduced amino acid sequences of the protein products revealed that SrTrxo2 specifically lacks the region corresponding to the α3-helix in SrTrxo1. Expression analysis of thermogenic and non-thermogenic S. renifolius tissues indicated that the SrTrxo1 and SrAOX transcripts are predominantly expressed together in thermogenic florets, whereas SrTrxo2 transcripts are almost undetectable in any tissue. Finally, functional in vitro analysis of recombinant SrTrxo1 and mitochondrial membrane fractions of thermogenic florets indicated its reducing activity on SrAOX proteins. Taken together, these results indicate that SrTrxo1 is likely to play a role in the redox regulation of SrAOX in S. renifolius thermogenic florets.

For the first time to our knowledge, we demonstrate that whole angiosperm individuals can survive gut passage through birds, and that this occurs in the field. Floating plants of the genus Wolffia are the smallest of all flowering plants. Fresh droppings of white-faced whistling duck Dendrocygna viduata ( n = 49) and coscoroba swan Coscoroba coscoroba ( n = 22) were collected from Brazilian wetlands. Intact Wolffia columbiana were recovered from 16% of D. viduata and 32% of Coscoroba samples (total = 164 plantlets). The viability of plants was tested, and asexual reproduction was confirmed. Wolffia columbiana is an expanding alien in Europe. Avian endozoochory of asexual angiosperm propagules may be an important, overlooked dispersal means for aquatic plants, and may contribute to the invasive character of alien species.